The present invention relates to an immobilized microbial consortium and a process for the preparation of the said immobilized microbial consortium, useful for rapid and reliable BOD estimation.
Rapid analytical devices have attracted tremendous interest and attention in science and technology for their wide range of possible application as an alternative to conventional analytical techniques. Analytical devices are sensitive to biological parameters and consist of a biological sensing element such as microbes, enzymes, etc., in close contact with a physico-chemical transducer such as an electrode, which converts biological signal to a quantitative response. These devices have several unique features such as compact size, simple to use, one step reagent-less analysis, low cost and quick real time results.
Rapid analytical devices, termed as biosensors, have the potential for a major impact in the human health care, environmental monitoring, food analysis and industrial process control. Among these, microbial biosensors (the devices using microbes as biological component), have great potential in environmental monitoring. Recent trends in biotechnology suggest that monitoring and control of pollutant by means of microbial biosensors may be of crucial importance. Such microbial sensors, constructed by entrapping the required micro-organisms in suitable polymeric matrices and attached to a transducer, function on the basis of assimilatory capacity of the micro-organisms. In addition, microbial biosensors are more stable and inexpensive for the determination of compounds of interest as compared to enzyme-based biosensors; where enzymes employed in enzyme-based biosensors require costly extraction and purification prior to use as biocatalysts. Further, micro-organisms employed in microbial biosensors show a high degree of stability as compared to enzymes.
The vast majority of micro-organisms are relatively easy to maintain in pure cultures, grow and harvest at low cost. Moreover, the use of microbes in biosensor field have opened up new possibilities and advantages such as ease of handling, preparation and low cost of the device. Such devices will help in monitoring the compounds of environmental interest such as Biochemical Oxygen Demand (BOD), heavy metals, pesticides, phenols, etc.
Among the environmental parameters, the potential demand for rapid BOD monitoring device is higher, since, BOD is a parameter which is measured most frequently by many industries for measuring the level of pollution of waste-waters. BOD provides information about the amount of biodegradable substances in waste-waters.
Conventional BOD test takes 3-5 days and as a consequence, is unsuitable for use in direct process control. A more rapid estimation of BOD is possible by developing a BOD biosensor. Such BOD biosensors are able to reduce the time of BOD test upto a great extent.
A number of microbial BOD sensors have been developed nationally and internationally (Rajasekar et al, 1992 and Karube, 1977). A number of pure cultures, eg., Trichosporon cutaneum, Hansenula anamola, Bacillus cereus, Bacillus subtilis, Klebsiella oxytoca, Pseudomonas sp., etc., individually, have been used by many workers for the construction of BOD biosensor (Preinenger et al, 1994; Hyun et al, 1993, Li and Chu 1991; Riedel et al, 1989 and Sun and Kiu, 1992). Karube et al, (1992) developed a BOD biosensor by utilizing thermophilic bacteria isolated from Japanese hot spring. On the other hand, most of the workers have immobilized activated sludge (Vanrolleghem et al 1990; Kong et al 1993; Vanrolleghem et al, 1984), or a mixture of two or three bacterial species (Iki, 1992 and Galindo et al 1992) on various membranes for the construction of BOD biosensor. The most commonly used membranes were polyvinyl alcohol, porous hydrophilic membranes, etc. Riedel et al, (1988), have used polyvinyl alcohol for the immobilization of Bacillus subtilis or Trichosporon cutaneum which are used for the development of BOD biosensor. Vinegar (1993) immobilized Klebsiella oxyoca on porous hydrophilic membranes such as nitrocellulose, acetyl cellulose, polyvinylidene flouride or polyether sulfone, 50-2000 micrometer thick. Cellulose acetate membrane was used for the immobilization of Lipomyces kononankoae and Asperillus niger (Hartmeier et al, 1993).
The drawback of such developed BOD biosensors which are constructed by using either single, pure culture or activated sludge is that they do not give reproducible results, as single microbe is not able to assimilate/degrade all the organic compounds and therefore may not respond for the total organic matter present in the test sample (eg., carbohydrates, proteins, fats, grease, etc.) Moreover, in the activated sludge either non-specific predominating microorganisms are present thereof or microorganisms with antagonistic effects are present which may produce erratic results. On the other hand, randomly selected mixtures of two or three micro-organisms also do not give reproducible, comparable BOD results. The reproducibility of the BOD biosensor can be obtained by formulating a defined microbial composition.
To avoid the discrepancies in BOD results as well as to get instant BOD values using rapid analytical devices, in the present invention, a defined microbial composition is formulated by conducting a systematic study, i.e., pre-testing of selected micro-organisms for use as a seeding material in BOD analysis of a wide variety of industrial effluents. The formulated microbial consortium is capable of assimilating most of the organic matter present in different industrial effluents. The formulated microbial consortium has been immobilized on suitable membrane i.e., charged nylon membrane useful for BOD estimation. Suitability of the charged nylon membrane lies in the specific binding between the negatively charged bacterial cell and positively charged nylon membrane. So, the advantages of the used membrane over other membranes are the dual binding i.e., adsorption as well as entrapment, thus resulting in a more stable immobilized membrane. Such specific microbial consortium based BOD analytical devices, may find great application in on-line monitoring of the degree of pollutional strength, in a wide variety of industrial waste-waters within a very short time (from 3-5 days to within an hour), which is very essential from pollution point of view.
For solving the aforementioned problems, the applicants have realized that there exists a need to provide a process for the preparation of a defined synergistic microbial consortium immobilized on a suitable support i.e., charged nylon membrane, useful for BOD estimation. The said microbial consortium is capable of assimilating most of the organic matter present in different industrial effluents.
The main object of the present invention is to provide a microbial consortium and a process for the preparation of the microbial consortium immobilized on a suitable support useful for BOD estimation.
The formulated microbial consortium comprises of cultures of the following bacteria viz., Aerornonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus and Enterobacter sakazaki. The individual bacteria of microbial consortium are pre-tested by using them as a seeding material in BOD analysis of a wide variety of industrial effluents. The micro-organisms have been selected for the formulation of microbial consortium on the basis of pre-testing. The formulated microbial consortium is obtained by inoculating a suspension of these bacteria individually. Incubating at 37xc2x0 C., mixing all bacterial cultures in equal proportions based on optical density and centrifuging. The resultant pellet is immobilized on suitable support, i.e., charged nylon membrane by entrapment and adsorption on the charged surface of the membrane. The said, charged immobilized microbial membrane has high viability, long stability and greater shelf-life as compared to the microbial consortium immobilized on conventional supports such as polyvinyl alcohol+nylon cloth.
Accordingly, another object of the present invention, is to provide a process for the production of immobilized formulated microbial consortium useful for monitoring the BOD load of a wide range of industrial effluents with low, moderate and high BOD load.
The present invention provides an immobilized microbial consortium and a process for the preparation of the said immobilized microbial consortium, useful for rapid and reliable BOD estimation of a wide range of industrial effluents with low, moderate and high BOD load.
The microbial consortium provided according to the present invention contains bacteria consisting of:
which facilitate the process of testing, giving BOD results of a wide variety of industrial effluents, performed at any place. Above microorganisms are deposited at Centre for Biochemical Technology Culture Collection (CBTCC) designated as stated above and will be made available to public on request as per normal official procedures.
The above micro-organisms are deposited with the American Type Culture Collection, Manasses, Va., USA and Deutsche Sammlung Von Mikroorganimen Und Zelikulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany on the dates and with designations as stated above.
The microbial consortium may contain the bacteria, in a preferred embodiment of the invention, in uniform amounts.
The microbial consortium of the present invention is useful for BOD estimation.
The bacterial cultures of the above microbial consortium are isolated from sewage. Sewage samples are collected from Okhla Coronation Plant near Okhla, New Delhi. Sewage is homogenized for 2 minutes and suspended in gram-negative nagative culture broth. Incubation is carried out for 24 hours. Cultures are plated on Mac Conkey""s agar. Colonies are mixed on a vortex mixer and all the cultures are isolated in pure form after several sub-cultures.
The immobilization technique of formulated microbial consortium of the present invention is carried out by inoculating the individual strains of the above mentioned bacteria separately in nutrient broth containing (per litre), 5.0 g peptic digest of animal tissue, 5.0 g of sodium chloride, 1.5 g of beef extract, 1.5 g yeast extract and 0.2 ml tween-80. All the cultures are incubated preferably at 37xc2x0 C. for approximately 16-24 hours in an incubator shaker. For gentle shaking, the incubator shaker is maintained at an appropriate rpm, preferably at 75 rpm. After sufficient growth is obtained, the bacterial cells from these individual cultures are taken in equal proportions based on optical density and then mixed for formulating microbial consortium. The resultant bacterial suspension is centrifuged at an appropriate rpm, preferably at 10,000 rpm for a period of 20 minutes. The resultant pellet is washed by dissolving in minimum quantity of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at an appropriate rpm, preferably at 10,000 rpm for a period of approximately 20 minutes. During centrifugation, the temperature is maintained preferably at 4xc2x0 C. The pellet thus obtained is immobilized on various membranes/supports such as charged nylon membrane and polyvinyl alcohol+nylon cloth.
For the immobilization of formulated microbial consortium on charged nylon membrane, the pellet of formulated microbial consortium is dissolved in 2 ml of phosphate buffer, 0.05M, pH 6.8 and filtered under vacuum. A number of immobilized microbial membranes are prepared under varying conditions of cell density and phase of cell growth. The immobilized microbial membranes thus obtained are left at room temperature for 4-6 hours to dry and stored at an appropriate temperature, preferably at 4xc2x0 C.
For immobilization of microbial consortium on polyvinyl alcohol (high molecular weight, i.e., 70,000 to 1,00,000 hot water soluble)+nylon cloth, a strip of nylon net (approx. 4xc3x974 inch2) is tightly bound to a glass plate with the help of an adhesive. The pellet of formulated microbial consortium is dissolved in 2.0 ml phosphate buffer, 0.05M, pH 6.8 and mixed with 2% polyvinyl alcohol (PVA). The mixture of PVA and culture is poured onto a tightly bound nylon net. The mixture is spread with the help of glass rod thoroughly. A PVA+nylon cloth membrane without microorganisms is also prepared simultaneously, for control. The prepared membranes are left at room temperature for 4-6 hours to dry and then stored at an appropriate temperature, preferably at 4xc2x0 C.
The immobilized microbial membranes thus obtained, are characterized with respect to cell density and phases of cell growth. For this, the individual microorganisms are grown for different time periods and a range of cell concentration is used for the immobilization on charged nylon membrane. The viability and stability of the immobilized microbial consortium is checked by storing at different pH and different temperatures. For checking the viability of immobilized microbial membranes, the membrane is placed on an agar plate in an inverted position and incubated at 37xc2x0 C. overnight. The colonies were observed for growth on agar plates. For the stability study, the prepared immobilized microbial membranes are stored at different temperatures i.e., 4xc2x0 C., 15xc2x0 C., 25xc2x0 C. and 37xc2x0 C. and different pH ranging from 6.4-7.2. The response of immobilized microbial membranes is observed at regular time intervals.
To enhance the sensitivity of the response, an amperometric system is designed using dissolved oxygen (DO) probe and a highly sensitive multimeter. An external source of xe2x88x920.65 volts is applied to the system to get the actual reduction of oxygen at cathode. A suitable polarization voltage i.e., xe2x88x920.65 volts between the anode and cathode selectively reduces oxygen at the cathode (Karube and Chang, 1991).
For the preparation of electrode assembly, the immobilized microbial membranes are sandwiched between an oxygen permeated teflon membrane and a porous membrane, i.e., cellulose acetate membrane. The immobilized microbial membrane is fixed directly onto the platinum cathode of an commercially available O2 probe.
The response characteristics of prepared immobilized microbial membranes is observed with synthetic sample i.e., glucose-glutamic acid (GGA), a reference standard used in BOD analysis. For this, the electrode assembly is dipped into a stirred PO4xe2x88x923 buffer solution. After a stable current was obtained, known strength of GGA was injected into the reaction assembly. Consumption of oxygen by the microbial cells immobilized on membrane caused a decrease in dissolved oxygen around the membrane. As a result, the values of dissolved oxygen decreased markedly with time until a steady state is reached. The steady state indicated that the consumption of oxygen by the immobilized microbial cells and the diffusion of oxygen from the solution to the membrane are in equilibrium. This value is recorded. Consumption of oxygen by the immobilized microorganisms is observed with multimeter in terms of current (nA). The change in current is linearly related to GGA standard over the range of 30 to 300 mg/l.
Accordingly, the invention provides a microbial consortium comprising a synergistic mixture of the following isolated bacterial present in equal proportions useful for rapid and reliable BOD estimation.
The invention further provides a process for the preparation of immobilized microbial consortium and the attachment of the same with an oxygen probe useful for the estimation of BOD load of a wide variety of industrial waste-waters, which comprises:
a) isolating a range of bacterial strains from sewage collected from sewage treatment plant;
b) culturing the said strains on nutrient media to get pure cultures;
c) testing the said individual pure bacterial cultures for use as seeding material in BOD analysis using glucose-glutamic acid (GGA) as a reference standard by recording BOD values exhibited by individual strains;
d) comparing the BOD values of the said bacterial strains with that of the observed BOD values using sewage as a seeding material collected from sewage treatment plant;
e) selecting the bacterial strains which have BOD values equal to or more than the BOD values of sewage as observed in step (d);
f) formulating the microbial consortium of selected bacterial strains obtained from step (e);
g) testing the formulated microbial consortium by comparing their BOD values with those of sewage used as a seeding material;
h) immobilizing the said formulated microbial consortium by inoculating bacterial strains individually, incubating the said bacterial strains, growing the said incubated strains and mixing them in equal proportions on the basis of optical density values;
i) centrifuging the resultant suspension to obtain pellets, washing the collected pellet by dissolving in PO4xe2x88x923 buffer, 0.025-0.075 M, pH 6.4-7.2, recentrifuging the pellet;
j) collecting the pellet from step (i), dissolving in 2.0-4.0 ml PO4xe2x88x923 buffer, 0.025-0.075 M, pH 6.4-7.2, to obtain cell slurry for cell immobilization;
k) filtering the obtained cell slurry on charged nylon membrane under vacuum for immobilization;
l) drying the immobilized microbial membrane obtained from step (k);
m) storing the dried immobilized microbial membrane obtained from step (l) preferably at 1-4xc2x0 C. in PO4xe2x88x923 buffer, 0.025-0.075 M, pH 6.4-7.2;
n) checking the viability of microorganisms in the said immobilized microbial membrane obtained from step(m);
o) attaching the immobilized microbial membrane obtained from step (m) with dissolved oxygen probe for the preparation of electrode assembly;
p) applying an external polarization voltage of xe2x88x920.65 V to the said electrode assembly obtained from step (o);
q) stabilizing the electrode assembly obtained from step (p) in PO4xe2x88x923 buffer, 0.025-0.075 M, pH 6.4-7.2, for 30-45 minutes;
r) observing the stability of the immobilized microbial membrane using stabilized electrode assembly obtained from step (q) by measuring the change in oxygen concentration in terms of current for BOD values covering a range of GGA concentrations;
s) characterizing the immobilized microbial membrane with respect to different variables, viz., cell density 100 xcexclxe2x88x921000 xcexcl, phase of cell growth 4 hours-16 hours, pH 6.4-7.2 and temperature 4xc2x0 C.-37xc2x0 C. in terms of response time using a range of GGA concentrations as in step (r);
t) selecting an appropriate immobilized microbial membrane from step (s) and attaching to an oxygen electrode as in step (o);
u) stabilizing the complete electrode assembly obtained form step (t) as in step (q);
v) testing the said stabilized electrode assembly by observing the change in oxygen concentration in terms of current for BOD values using a range of industrial effluents ranging from 0.05%-20.0% covering low, moderate and high biodegradable effluents. The change in current being linearly proportional to the amount of biodegradable organic matter present in the effluent.
In an embodiment of the present invention, the formulated microbial corsortium is obtained by inoculating a suspension of the bacteria selected from a group consisting of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus and Enterobacter sakazaki. 
In another embodiment of the present invention, the individual strains of the above mentioned bacteria are inoculated separately in a nutrient broth.
In a further embodiment of the present invention, the incubation of bacterial strains is carried out by gentle agitation at approximately 75-100 rpm.
In one of the embodiment of the present invention, the growth of incubated bacterial strains is carried out at a temperature ranging between 30-37xc2x0 C. for a period of 16-24 hours.
In an embodiment of the present invention, the said individual strains are mixed in equal proportions.
In a further embodiment of the present invention, the resultant microbial consortium is centrifuged at appropriate rpm preferably at 8,000-12,000 rpm for a period of approximately 20-30 minutes at a temperature ranging from 1-4xc2x0 C.
In another embodiment of the present invention, the resultant pellet is washed by dissolving in an appropriate quantity of PO4xe2x88x923 buffer, 0.025-0.075 M, pH 6.4-7.2 and recentrifuged at an approximate rpm in the range 8,000-12,000 rpm at a temperature preferably at 4xc2x0 C.
In an embodiment of the present invention, the resultant cell pellet obtained is immobilized by dissolving in 1.0-2.0 ml of phosphate buffer ranging between 0.025-0.075 M, pH 6.4-7.2 to obtain cell slurry.
In one of the embodiment of the present invention, the resulting cell slurry is filtered on charged nylon membrane under vacuum.
In an embodiment of the present invention, the immobilized microbial membrane is dried at appropriate temperature, ranging between 25-35xc2x0 C., for a period ranging between 4-6 hours.
In a further embodiment of the present invention, the dried immobilized membrane is stored in phosphate buffer, 0.05M, pH 6.8 at appropriate temperature ranging between 1-4xc2x0 C.
In one of the embodiment of the present invention, the prepared immobilized microbial membrane is placed on nutrient agar plate and incubated at temperature ranging between 30xc2x0 C.-37xc2x0 C. for a period of 16-24 hours to observe the bacterial growth for viability of immobilized microorganisms.
The invention further provides a method for the estimation of BOD which comprises of an immobilized microbial membrane.
In one of the embodiment of the present invention, the dried immobilized microbial membrane is attached to dissolved oxygen probe with O ring for the preparation of electrode assembly.
In an embodiment of the present invention, the stability of the immobilized microbial membrane stored at different temperatures ranging from 4xc2x0 C.-37xc2x0 C. was observed using electrode assembly. The response was observed in terms of change in current.
In another embodiment of the present invention, the stable and viable immobilized microbial membrane was used for rapid and reliable BOD analysis using GGA as a reference standard in the concentration range of 30-300 mg/l.
In a further embodiment of the present invention, the immobilized microbial membrane was used for rapid and reliable BOD analysis of industrial effluents ranging from low, moderate to high biodegradable organic matter.